Hexavalent chromium is cytotoxic and genotoxic to American alligator cells
Introduction
The American alligator (Alligator mississippiensis) is a long lived, apex predator inhabiting primarily coastal areas of the southeastern US. Once listed as endangered due to overhunting, careful conservation efforts led to delisting in 1987. Localized populations of alligators are exposed to environmental contaminants through a variety of sources. Development in and near their ecosystem can lead to chemical and agricultural runoff. Since the alligator populations have successfully recovered after being listed as endangered in the 1970s, they can be used as a suitable monitor of environmental pollution (Delany et al., 1988).
Chromium (Cr) has recently been shown to be a metal of global concern (Wise et al., 2009). Hexavalent chromium (Cr(VI)) is a known human carcinogen and can damage DNA and impair reproduction and development (Al-Hamood et al., 1998, Bataineh et al., 1997, Chowdhury and Mitram, 1995, Holmes et al., 2008, IARC, 1990, Mancuso, 1997, Wise et al., 2008a, Wise et al., 2008b, Witmer et al., 1989). A few studies have measured Cr in alligators and reveal a concern. A study of alligators in South Carolina found a cluster of alligators had relatively high concentrations of Cr in liver tissue with several animals having levels over 30 μg/g (Campbell et al., 2010). Horai et al. (2014) showed that Cr accumulates in adult alligators based on comparisons between juvenile and adult alligator livers at 3 different sites in Florida. Interestingly of the three sites tested the Cr levels in adult alligators from Merritt Island National Wildlife Refuge (MINWR) were the highest and were 3 times higher than the other two sites suggesting localized pollution (Horai et al., 2014).
However, while these studies show alligators may be exposed to significant levels of Cr, no studies have considered potential adverse effects as a result of Cr exposure in alligators. In fact, consideration of the available literature shows Cr is poorly studied in aquatic reptiles and appears to be limited to two reports. One study, in green sea turtle cells, found Cr(VI) to be one of the most cytotoxic of four metals tested (Tan et al., 2010). The other considered hawksbill sea turtle cells and found both particulate and soluble Cr(VI) were cytotoxic and clastogenic (Wise et al., 2014).
The explanation for the lack of data is presumably due to the lack of access to experimental models of aquatic reptiles. Many species are endangered and protected. However, it is possible to gain important species-specific insights into potential toxicological impacts through aquatic reptile cell cultures. Accordingly, to begin developing a better understanding of pollution impacts on alligators and crocodilians in general, we investigated the cytotoxicity and genotoxicity of chromium in fibroblasts developed from American alligator scute tissue. Because the major health concern in the environment is the hexavalent form of chromium, and since particulate Cr(VI) is considered to be a more potent carcinogen than soluble Cr(VI) (IARC, 1990, Wise et al., 2008a, Wise et al., 2008b), we focused our study on particulate and soluble Cr(VI) compounds.
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Materials
All plasticware was purchased from BD Falcon. Dulbecco’s Phosphate-Buffered Saline (DPBS), RPMI media with Glutagro was purchased from orning. Potassium chloride, demecolcine, lead chromate, and sodium chromate were purchased from Sigma/Aldrich. Crystal violet, methanol and acetic acid were purchased from JT Baker. Microscope slides were purchased from Thermo Scientific. Giemsa stain was manufactured by Rica Chemical Co. Fetal Bovine Serum (FBS), Gurr’s buffer, trypsin, penicillin-streptomycin
Cytotoxicity
There was a concentration-dependent decrease in alligator cell survival after both lead chromate and sodium chromate treatment compared to the untreated controls. Treatments of 0.1, 0.5, 1, and 5 μg/cm2 lead chromate for 24 h induced 93, 70, 60, and 1% relative survival, respectively (Fig. 1). Treatments of 0.5, 1, 2.5, and 5 μM sodium chromate induced 87, 78, 60, and 25% relative survival, respectively (Fig. 2). The estimated LC50s for lead chromate and sodium chromate were 2.1 μg/cm2 (95%
Discussion
Our data show particulate and soluble Cr(VI) are cytotoxic to alligator cells. Our study is the first to report cytotoxicity in alligator cells and one of only a few to report cytotoxicity in any reptilian species. Our data are consistent with previous cytotoxicity studies in hawksbill sea turtle, loggerhead sea turtle, and green sea turtle cells (Tan et al., 2010, Wang et al., 2013, Webb et al., 2014, Wise et al., 2014). Three of these studies considered metal cytotoxicity (Tan et al., 2010,
Acknowledgements
The authors would like to thank Matt Guillette for assistance in collecting alligator samples; Therry The and Christy Gianios, Jr. for technical support. Research reported in this publication was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number R01ES016893 (JPW). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Additional
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- 1
Present address: Wise Laboratory of Environmental and Genetic Toxicology, Department of Pharmacology and Toxicology, University of Louisville, 505 S. Prescott St, Louisville, KY 40292, USA.
- 2
Present address: Toxikon Corp, 15 Wiggins Avenue, Bedford, MA 01730, USA.